1
J. Phyaiol. (1979), 290, pp. 1-10 With 4 text-figures Printed in Great Britain
THE EFFECTS OF TENOTOMY UPON THE CONTRACTION CHARACTERISTICS OF MOTOR UNITS IN RABBIT SOLEUS MUSCLE
BY J. BAGUST* From the Department of Physiology, University of Bristol, Bristol, BS8 1TD
(Received 18 September 1978) SUMMARY
1. Isometric contractions of tenotomized rabbit solei have been compared with a group of unoperated control muscles. A large decrease in the tension developed by the tenotomized muscle was accompanied by a slight shortening of the twitch contraction time, and a larger, progressive, reduction in the time to half relaxation. 2. A comparison of motor units obtained from muscles which had been tenotomized for six weeks with those of the control muscles showed a large reduction in the range of contraction and half relaxation times of the units from the tenotomized muscles. 3. The mean motor unit tension (expressed as a percentage of the whole muscle tension) was similar for both those units from the control muscles and those from muscles which had been tenotomized for six weeks, indicating a uniform atrophy of motor units within the tenotomized muscles. 4. It was concluded that the change in the pattern of motor unit contraction times was not the result of a process of differential atrophy favouring the preservation of the faster contracting motor units. 5. A correlation between axon conduction velocity and both the speed of contraction and the size (tension) of the motor units was demonstrated in the control muscles. Following tenotomy the relationship between axon conduction velocity and motor unit tension was lost. INTRODUCTION
The effect of tenotomy upon the slow-twitch rabbit soleus muscle has been shown to be a rapid atrophy accompanied by an increase in both the speeds of contraction and relaxation of the muscle twitch (Vrbova, 1963; Buller & Lewis, 1965; Salmons & Vrbova, 1969). This change in the time course of the twitch contraction has been used as evidence in support of the claim that the contractile speed of mammalian muscles is determined by the pattern of nervous activity reaching them (Salmons & Vrbova, 1969). However Buller & Lewis (1965) suggest that the effects of tenotomy seen in rabbit soleus might result from a greater degree of atrophy occurring in those muscle fibres with the slowest time courses of contraction, rather than a change in their speeds of contraction. * Supported by the Muscular Dystrophy Group of Great Britain. Present address: Department of Neurophysiology, Southampton University, Southampton S09 3TU.
0022-3751/79/3160-0742 $01.50 © 1979 The Physiological Society I
PHY
2
J. BAGUST This explanation presupposes the muscle to contain a mixture of fast and slowly contracting fibres. As yet no studies on the contractile properties of single mammalian muscle fibres have been reported, but it is assumed to be possible to obtain an indication of the spectrum of fibre contractile speeds by an examination of the properties of the individual motor units within a muscle. An initial study of the motor unit composition of rabbit soleus confirmed that the normal muscle contained motor units having widely different contraction times and so a further investigation was made to look for evidence of differential atrophy between fast and slowly contracting motor units following tenotomy. METHODS
In order to allow the direct comparison of results from different experiments the rabbits used were chosen to have body weights close to 3 kg (mean = 2-8 kg, S.D. = 0-22 kg, n = 15 for the control animals). Most were cross bred Californian of either sex, although some of the control rabbits were New Zealand Whites. In the initial experiments the animals were anaesthetized with a mixture of pentobarbitone sodium (Nembutal, 7-5 mg/ml.) and urethane (250 mg/ml.) given intravenously. However in later experiments it was found that the careful use of Nembutal alone (20 mg/ml.), administered intravenously, gave better control over the depth of anaesthesia. The dissecting and recording conditions were similar to those described for cat soleus motor units (Bagust, 1974). The dual blood supply to the rabbit soleus described by Buller & Lewis (1965) necessitated careful dissection of those vessels entering soleus from the overlying gastrocnemius muscle, so that the latter muscle could be removed without endangering the soleus blood supply. As in the cat studies it was possible to change the tension transducers with the minimum of disturbance to the muscle, allowing the recording of the smallest motor unit twitch in a tenotomized muscle, and both twitches and tetani of whole normal muscles. Three different strain gauges were used; a Statham GI-81, linear up to 49N; an Ether UFl-16, linear up to 7N; and a high sensitivity Ether UFI-2, linear up to 0 7 N. Motor units were isolated by splitting filaments from the 7th lumbar and 1st sacral ventral roots, exposed by a dorsal laminectomy. The signals from the strain gauge were amplified and fed into a Moldular One computer (Computer Technology Ltd) via an 11 bit analogue to ditigal converter, for on-line analysis. This enabled several twitches to be averaged where necessary, to increase the signal to noise ratio. Full length-tension curves for whole muscle twitches and tetani were plotted at the beginning and end of each experiment. These were similar in shape to those shown for cat soleus muscles by Buller & Lewis (1963) and Bagust (1974). The length-tension patterns of individual rabbit soleus motor units were similar to those of the parent muscle. (The mean difference between the muscle lengths for optimum tension development of 31 motor units and their six parent muscles was -0-1 mm, S.D. 1-2 mm for twitches and -0-5 mm, S.D. 1I- mm for tetani). Both whole muscle and motor unit tetani had optima at lengths shorter than those of the twitches, (- 17 mm, S.D. 0-49 mm for the whole muscle tetani and - 2-0 mm, S.D. 0-96 mm for motor unit tetani). Because of the similarity in the length-tension properties between the whole muscles and their motor units, full length-tension curves were plotted for each of the whole muscles, but the motor units were examined only around their parent muscle optima. This enabled larger samples of motor units to be taken from each muscle. The temperature of the paraffin pools surrounding the leg and back dissections was maintained at 37 + 1 TC by radiant heat from car headlamps controlled by a feed-back circuit from probes in the pool. This was particularly important because it was noticed that changes in temperature had different effects upon the tensions developed by normal and tenotonized muscles. In both cases an increase in temperature resulted in a shortening of the twitch time to peak, but in the unoperated muscle this was accompanied by an increase in the twitch tension (cf. cat soleus, Buller, Ranatunga & Smith, 1968) whereas those muscles which had been tenotomized for more than 40 days exhibited a fall in both twitch and tetanus tension.
3
MOTOR UNITS IN TENOTOMIZED RABBIT SOLEUS
Aseptic tenotomies were performed under light pentobarbitone anaesthesia supplemented by 1 % procaine injected subcutaneously around the ankle joint. All the tendons around the right ankle were sectioned, care being taken to avoid damage to the main blood vessels and nerves. Following recovery the animals were kept in pens on a solid floor for a week before being transferred to wire cages. A
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Days Days in of twitch Fig. 1. The time course the changes (A) tension, (B) twitch-tetanus ratio, (C) time to peak and (D) time to half relaxation, of rabbit soleus following tenotomy. The continuous horizontal lines represent the mean value and the interrupted lines the mean + S.D. of the fifteen control muscles. Each point represents one tenotomized muscle, the open circles show the four muscles in which motor units were examined.
The terminal experiments were performed on twenty-six animals between 4 and 62 days after tenotomy. Before the animal was anaesthetized an attempt was made to determine if there was any significant tendon regeneration by observing the locomotion of the animals and feeling through the skin for any stretching of the leg muscles when the foot was flexed. In only two cases was it felt that there might have been significant tendon regrowth, and both of these showed less muscular atrophy than might have been expected. These muscles were not used for the motor unit studies. RESULTS
1. The effects of tenotomy upon whole muscle contraction characteristics The tension developed in response to maximal stimulation of the motor nerve fell rapidly in the first 20 days following tenotomy (Fig. 1 A). The tetanus tension declined faster than that of the twitch, resulting in a progressive increase of the twitch-tetanus ratio with time after tenotomy (Fig. 1 B). (The twitch-tetanus ratios in Fig. 1 B and Table 1 are the maximum values recorded. These seldom occurred at the muscle optimum length for either twitches or tetani and so do not correspond to the ratios of the twitch and tetanus tensions in Table 1.) The tetanic response of muscles which had been tenotomized for over 40 days often fell to a plateau after reaching an initial peak of tension (Fig. 2). This was accompanied by a reduction in the amplitude of the electromyogram recorded from the surface of 1
1-2
4
J. BAGUST the muscle suggesting the failure of neuromuscular transmission on repetitive stimulation. This interpretation was confirmed by direct stimulation of the muscle after treatment with curare when the fall in tension was abolished (Fig. 2D). (The experiments involving curare were performed in collaboration with D. M. Lewis). No TABLE 1. The mean isometric contraction properties of the fifteen control solei and the eighteen muscles that had been tenotomized for 20 days or longer. The significance of the differences between the mean values for the two groups is shown in the last column, calculated by Student 's t test (n.s. = difference between the means is not significant, P > 0 05)
Twitch tension(N) Tetanus tension(N) Twitch/tetanus Time to peak (msec) Half relaxation time (msec)
Control
Tenotomized
n mean S.D. 15 3-6 0*9 15 16-2 2-0 15 0-21 004 15 50-8 11*3 15 83-3 32*5
n mean S.D. 18 0*5 0-1 18 1.5 04 18 045 0-11 18 46*7 7-2 18 52*8 14*2
A
P < 0.001 < 0-001 < 0X001 n.s. < 0*001
B
05N
iON
100 msec
100 msec
C
D
05N [J
N 3-0
(i 100 msec
11111
111 _ 100 msec
Fig. 2. Isometric myograms recorded from tenotomized rabbit solei. A, single twitch response from a muscle tenotomized for 33 days; B, tetanic response (80 Hz 300 msec) of the same muscle as in A, superimposed upon a single twitch; C, tetanic response (80 Hz 300 msec) of a soleus muscle that had been tenetomized for 57 days; underneath the mechanical record is the whole muscle electromyogram recorded simultaneously. D, titanic response (80 Hz 300 msec) of a 47 day tenotomized soleus, stimulated indirectly through the nerve (lower record) and directly, after curare (upper record).
5 MOTOR UNITS IN TENOTOMIZED RABBIT SOLEUS attempt was made at systematically quantifying the extent of this neuromuscular failure, but it seems probable that it was an important factor in the increased twitchtetanus ratio. In Table 1 the contraction properties of those muscles examined 20 days or more after tenotomy are compared with corresponding values for the control muscles. The effect of tenotomy upon the time course of the muscle twitch contraction was a non-significant decrease in the time to peak (Fig. 1 C) and a progressive increase in the rate of relaxation as measured by the time to half relaxation (Fig. 1 D). Each of the muscles which had been tenotomized for 50 days or more had a time to half relaxation that was less than half that of the mean control value of 83-3 msec. The greater degree of atrophy of soleus compared to its neighbouring fast-twitch muscles reported by previous authors (McMinn & Vrbova, 1962) was confirmed in these experiments. When compared with muscles from five unoperated animals the mean weight (g/kg body wt.) of solei from thirteen animals which had been tenotomized for more than 20 days, was only 48% ( ± 3 %) of the control value for the tenotomized muscle whereas the corresponding solei from the unoperated side of these animals weighed 109 % ( ± 5%) of the control value. The tenotomized gastrocnemi averaged 67 % ( ± 2 %) of the control value, compared to 107 % ( ± 3 %) for the unoperated muscles, and the corresponding values for plantaris were 67 % ( ± 5 %) and 99 % (± 4 %) respectively. There was little evidence for hypertrophy of the contralateral, unoperated, muscle induced by tenotomy of the right leg.
2. The effect of tenotomy upon the properties of rabbit soleus motor units The control population of ninety-five motor units was isolated from fifteen soleus muscles from unoperated rabbits (sample size from three to ten motor units per experiment). Eight-one motor units were also isolated from ,four of the twenty-six tenotomized rabbit solei approximately six weeks (39, 41, 43 and 45 days) after sectioning all the tendons around the right ankle. These muscles are indicated in Fig. 1 by the open symbols. Because it was not possible to measure the degree of neuromuscular fatigue affecting any individual motor unit, comparison of motor unit contractile strengths between tenotomized and control muscles had to be confined to a consideration of twitch tensions. This might have been expected to result in an underestimate of motor unit size due to the damping effect of the series elastic element. However, the mean value for the 95 control motor unit twitch tensions, when expressed as a percentage of the whole muscle twitch tension, was very close to the corresponding value for the tetanus tensions (mean tetanus tension = *01 0/, S.D. 0-52 %, mean twitch tension = 1.00 %, S.D. 0-54 %/ t = 0.2), suggesting that the errors caused by using twitch tensions to estimate motor unit size were slight. Following tenotomy the mean motor unit twitch tension fell to less than 10% of the mean value for the control units (Table 2). In order to allow comparison of the control motor units with those from the tenotomized muscles the histograms of Fig. 3 have been plotted with each motor unit tension expressed as a percentage of the corresponding whole muscle tension. When treated in this manner no significant difference was found between motor units from the control and tenotomized muscles in either their mean values or in their distributions (Table 2).
J. BAG UST
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7 MOTOR UNITS IN TENOTOMIZED RABBIT SOLEUS The effect of tenotomy upon the time course of the motor unit twitch contractions was a shortening of both the mean time to peak and time to half relaxation but the histograms of Fig. 3 show that this was not a uniform speeding up of the motor units. The range of motor unit contraction and half relaxation times in the control muscles 25
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Fig. 3. Histograms showing the distributions of isometric contraction characteristics of motor units from both the control and tenotomized muscles. A, twitch tensions of the ninety-five motor units from control muscles expressed as a percentage of the whole muscle tension. B, twitch tensions of the eighty-one motor units from tenotomized muscles expressed as a percentage of the whole muscle tension. C and D, control and tenotomized muscle motor unit times to peak and E and F, times to half relaxation.
was wide, from 22 to 123 msec for the time to peak, and 22-247 msec for the half relaxation times. In the case of the motor units from the tenotomized muscles the slower end of the population was missing. No unit having a time to peak greater than 70 msec, or a half relaxation time greater than 100 msec was found whereas 35 % of the motor units from the control muscles had contraction times longer than 70 msec. These units contributed 21*4 % to the total population tension. At the other end of the spectrum there was no evidence for an increase in the speed of contraction of the faster motor units; the fastest motor unit examined in the tenotomized muscles (contraction time 27 msec, half relaxation time 36 msec) was slower than the fastest control muscles motor unit.
J. BAGUST Possible relationships between axon conduction velocity and motor unit contraction time or tetanus tension were investigated using the method of least squares. Linear regressions were calculated for the ten control experiments in which more than five motor units were isolated. When each experiment was considered individually the 8
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_0 0 40 80 60 80 Conduction velocity (m/sec) Fig. 4. Mechanical responses of a control rabbit soleus muscle and four motor units from that muscle. At the beginning of each trace is a calibration pulse, and the bar under each represents 100 msec. A, whole muscle twitch; B, whole muscle tetanus (80 Hz 300 msec) superimposed on a single twitch response. C-F, single motor unit twitches. G and H. plot of antidromic axon conduction velocity against (G) motor unit time to peak and (H) tetanus tension of ten motor units from a single muscle. The regression lines were fitted by the method of least squares.
040 __
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value of the correlation coefficient (r) exceeded the 5 % significance level (P < 0.05) in eight of the ten cases for the regression of contraction time on conduction velocity, and in five out of ten cases for the regressions of tetanus tension on conduction velocity. Fig. 4 shows plots of motor unit contraction times and tetanus tensions against axon antidromic conduction velocities for the control experiment in which the largest number of motor units were examined. When all ninety-five control motor units were treated as one group the significance of the correlation coefficients increased to the 0.1 % level, (regression of contraction time on conduction velocity r = 0 447, regression of tetanus tension on conduction velocity r = 0-471). Following tenotomy significant values for the correlation coefficient were found for the regression of motor unit time to peak upon conduction velocity in two of the four experiments, and when all eighty-one units were treated as a single group there was a high level of correlation (r = 0-366, P < 0.01). Similar treatment of the regression of
9 MOTOR UNITS IN TENOTOMIZED RABBIT SOLEUS motor unit twitch tension upon conduction velocity revealed no significant correlations in either individual experiments or pooled data. In addition to the changes occurring in the contractile characteristics of the motor units following tenotomy evidence was also found for differences in the conduction properties of the nerve axons supplying motor units from control and tenotomized muscles. The mean antidromic conduction velocity increased from a value of 620 m/sec in the control animals to 74*3 m/sec in the tenotomized animals, a difference of 19*8 % which was significant at the 1 % level (Table 2). This observation was not investigated further. DISCUSSION
The results reported here confirm those of Buller & Lewis (1965) that following teneotomy there is a rapid decrease in the ability of the rabbit soleus muscle to develop tension, and that this is accompanied by a slight shortening of the time to peak of the twitch and a larger reduction in the time to half relaxation. They suggest that these changes might be the result of a more rapid atrophy of the slowly contracting fibres within a muscle containing fibres of different contractile speeds, rather than a change in the speed of the contractile elements themselves. This view receives support from the observation that following tenotomy the soleus muscle atrophies faster than its neighbouring fast twitch muscles (rabbit, McMinn & VrbovA 1962; guinea-pig, Tomanek & Cooper 1972). The wide range of motor unit contraction times found in the normal rabbit soleus, and the apparent loss of all the slower motor units in the tenotomized muscles are quite compatible with the concept of differential atrophy. However, if a significant number of motor units within a muscle ceased to function entirely then the remaining units would each contribute a larger fraction to the total muscle tension. The observed loss of the 35 % of the more slowly contracting units from the normal muscle (contributing 21 % of the muscle tension) would be expected to increase the mean motor unit contribution to the whole muscle tension from 1 00 % in the normal muscle to 1*25 % in the tenotomized muscle, but there was no significant change in the mean motor size as measured by the percentage tension, nor any change in the pattern of distribution of tensions between motor units from the control and tenotomized muscles. It is therefore unlikely that a differential atrophy of motor units is responsibe for the changes in muscle contraction characteristics following tenotomy. The possibility of a differential atrophy of muscle fibres within a motor unit has not been excluded by these experiments. The loss of slowly contracting fibres from motor units composed of fibres widely varying in contractile speeds might produce the observed results. Although no direct recordings of the contractile properties of individual muscle fibres within motor units have been made Edstr6m & Kugelberg (1968) have demonstrated a high degree of histochemical uniformity amongst muscle fibres of the same motor unit in the anterior tibial muscle of the rat, suggestive evidence for mechanical uniformity of muscle fibres within motor units. The increase in the mean antidromic conduction velocity of the motor unit axons 40 days after tenotomy was similar to increases in nerve conduction velocities that have been reported by other workers using rabbits in experiments lasting several weeks (Quillam, 1958; Cragg & Thomas, 1964). It therefore seems most probable that this represents an ageing process and is unrelated to the effects of tenotomy. It is
J. BAGUST possible that the differences in motor unit contractile speeds between the control and tenotomized muscles were also an effect of ageing and unrelated to tenotomy. If the correlation between conduction velocity and motor unit time to peak is taken to indicate a causal relationship the increase in antidromic conduction velocity would be expected to result in an increase in the speed of contraction of the motor units, as was found in these experiments. There is no information available about the effects of age on the contractile speeds of motor units in the rabbit soles, but in the cat soleus (a more homogeneus muscle than the rabbit soleus) the mean motor unit time to peak has been shown to increase rather than decrease between six weeks of age and adult (Bagust, Lewis & Westerman, 1974; Bagust, 1974). These experiments were designed to examine the role of the motor unit in the changes taking place in the rabbit soleus muscle following tenotomy. They have shown that a process of differential atrophy of motor units is unlikely to be the explanation for the observed changes in the time course of the twitch contraction, and a change in the contractile elements themselves remains a possibility. They do not give any indication of the factors altered by tenotomy that might result in such changes, except to show that the effects are widespread, involving not only the speed of muscle contraction, but also neuromuscular transmission and even the effects of temperature changes on the twitch tension, and must be interpreted with caution. 10
I would like to thank Dr D. M. Lewis and Professor A. J. Buller for their help and advice. This work was supported by the Muscular Dystrophy Group of Great Britain and the computer used was purchased by a grant from the Medical Research Council.
REFERENCES BAGUST, J. (1974). Relationships between motor nerve conduction velocities and motor unit contraction characteristics in a slow twitch muscle of the cat. J. Physiol. 238, 269-278. BAGUST, J., LEWIS, D. M. & WESTERMAN, R. A. (1974). The properties of motor units in a fast and a slow twitch muscle during post-natal development in the kitten. J. Phy8iol. 237, 75-90. BULLER, A. J. & LEWIS, D. M. (1963). Factors affecting the differentiation of mammalian fast and slow muscle fibres. In The Effect of Use and Disuse on Neuromuscular Functions, ed. GUTMANN, E. & HNIK, P., pp. 149-159. Prague, Amsterdam: Elsevier. BULLER, A. J. & LEWIS, D. M. (1965). Some observations on the effects of tenotomy in the rabbit. J. Physiol. 178, 326-342. BULLER, A. J., RANATUNGA, K. W. & SMIrH, JANET M. (1968). The influence of temperature on the contractile characteristics of mammalian fast and slow twitch skeletal muscles. J. Physiol. 196, 82P. CRAGG, B. G. & THOMAS, P. K. (1964). The conduction velocity of regenerated peripheral nerve fibres. J. Physiol. 171, 164-175. EDSTROM, L. & KUGELBERG, E. (1968). Histochemical composition distribution of fibres and fatigability of singlemotor units. Anterior tibial muscle of the rat. J. Neurosurg. Psychiat. 31, 424-433. McMINw, R. M. H. & VRBOvA, G. (1962). Morphological changes in red and pale muscles following tenotomy. Nature, Lond. 195, 509. QUILLIAM, T. A. (1958). Growth changes in sensory nerve fibre aggregates undergoing remyelination. J. Anat. 92, 383-398. SALMONS, S. & VRBOVA, G. (1969). The influence of activity on some contractile characteristics of mammalian fast and slow muscles. J. Physiol. 201, 535-549. TOMANEK, R. J. & COOPER, R. R. (1972).Ultrastructural changes in tenotomized fast and slow-twitch muscle fibres. J. Anat. 113, 409-424. VRBOVk, G. (1963). The effect of motoneurone activity on the speed of contraction of striated muscle. J. Physiol. 169, 513-526.
J. Phyaiol. (1979), 290, pp. 1-10 With 4 text-figures Printed in Great Britain
THE EFFECTS OF TENOTOMY UPON THE CONTRACTION CHARACTERISTICS OF MOTOR UNITS IN RABBIT SOLEUS MUSCLE
BY J. BAGUST* From the Department of Physiology, University of Bristol, Bristol, BS8 1TD
(Received 18 September 1978) SUMMARY
1. Isometric contractions of tenotomized rabbit solei have been compared with a group of unoperated control muscles. A large decrease in the tension developed by the tenotomized muscle was accompanied by a slight shortening of the twitch contraction time, and a larger, progressive, reduction in the time to half relaxation. 2. A comparison of motor units obtained from muscles which had been tenotomized for six weeks with those of the control muscles showed a large reduction in the range of contraction and half relaxation times of the units from the tenotomized muscles. 3. The mean motor unit tension (expressed as a percentage of the whole muscle tension) was similar for both those units from the control muscles and those from muscles which had been tenotomized for six weeks, indicating a uniform atrophy of motor units within the tenotomized muscles. 4. It was concluded that the change in the pattern of motor unit contraction times was not the result of a process of differential atrophy favouring the preservation of the faster contracting motor units. 5. A correlation between axon conduction velocity and both the speed of contraction and the size (tension) of the motor units was demonstrated in the control muscles. Following tenotomy the relationship between axon conduction velocity and motor unit tension was lost. INTRODUCTION
The effect of tenotomy upon the slow-twitch rabbit soleus muscle has been shown to be a rapid atrophy accompanied by an increase in both the speeds of contraction and relaxation of the muscle twitch (Vrbova, 1963; Buller & Lewis, 1965; Salmons & Vrbova, 1969). This change in the time course of the twitch contraction has been used as evidence in support of the claim that the contractile speed of mammalian muscles is determined by the pattern of nervous activity reaching them (Salmons & Vrbova, 1969). However Buller & Lewis (1965) suggest that the effects of tenotomy seen in rabbit soleus might result from a greater degree of atrophy occurring in those muscle fibres with the slowest time courses of contraction, rather than a change in their speeds of contraction. * Supported by the Muscular Dystrophy Group of Great Britain. Present address: Department of Neurophysiology, Southampton University, Southampton S09 3TU.
0022-3751/79/3160-0742 $01.50 © 1979 The Physiological Society I
PHY
2
J. BAGUST This explanation presupposes the muscle to contain a mixture of fast and slowly contracting fibres. As yet no studies on the contractile properties of single mammalian muscle fibres have been reported, but it is assumed to be possible to obtain an indication of the spectrum of fibre contractile speeds by an examination of the properties of the individual motor units within a muscle. An initial study of the motor unit composition of rabbit soleus confirmed that the normal muscle contained motor units having widely different contraction times and so a further investigation was made to look for evidence of differential atrophy between fast and slowly contracting motor units following tenotomy. METHODS
In order to allow the direct comparison of results from different experiments the rabbits used were chosen to have body weights close to 3 kg (mean = 2-8 kg, S.D. = 0-22 kg, n = 15 for the control animals). Most were cross bred Californian of either sex, although some of the control rabbits were New Zealand Whites. In the initial experiments the animals were anaesthetized with a mixture of pentobarbitone sodium (Nembutal, 7-5 mg/ml.) and urethane (250 mg/ml.) given intravenously. However in later experiments it was found that the careful use of Nembutal alone (20 mg/ml.), administered intravenously, gave better control over the depth of anaesthesia. The dissecting and recording conditions were similar to those described for cat soleus motor units (Bagust, 1974). The dual blood supply to the rabbit soleus described by Buller & Lewis (1965) necessitated careful dissection of those vessels entering soleus from the overlying gastrocnemius muscle, so that the latter muscle could be removed without endangering the soleus blood supply. As in the cat studies it was possible to change the tension transducers with the minimum of disturbance to the muscle, allowing the recording of the smallest motor unit twitch in a tenotomized muscle, and both twitches and tetani of whole normal muscles. Three different strain gauges were used; a Statham GI-81, linear up to 49N; an Ether UFl-16, linear up to 7N; and a high sensitivity Ether UFI-2, linear up to 0 7 N. Motor units were isolated by splitting filaments from the 7th lumbar and 1st sacral ventral roots, exposed by a dorsal laminectomy. The signals from the strain gauge were amplified and fed into a Moldular One computer (Computer Technology Ltd) via an 11 bit analogue to ditigal converter, for on-line analysis. This enabled several twitches to be averaged where necessary, to increase the signal to noise ratio. Full length-tension curves for whole muscle twitches and tetani were plotted at the beginning and end of each experiment. These were similar in shape to those shown for cat soleus muscles by Buller & Lewis (1963) and Bagust (1974). The length-tension patterns of individual rabbit soleus motor units were similar to those of the parent muscle. (The mean difference between the muscle lengths for optimum tension development of 31 motor units and their six parent muscles was -0-1 mm, S.D. 1-2 mm for twitches and -0-5 mm, S.D. 1I- mm for tetani). Both whole muscle and motor unit tetani had optima at lengths shorter than those of the twitches, (- 17 mm, S.D. 0-49 mm for the whole muscle tetani and - 2-0 mm, S.D. 0-96 mm for motor unit tetani). Because of the similarity in the length-tension properties between the whole muscles and their motor units, full length-tension curves were plotted for each of the whole muscles, but the motor units were examined only around their parent muscle optima. This enabled larger samples of motor units to be taken from each muscle. The temperature of the paraffin pools surrounding the leg and back dissections was maintained at 37 + 1 TC by radiant heat from car headlamps controlled by a feed-back circuit from probes in the pool. This was particularly important because it was noticed that changes in temperature had different effects upon the tensions developed by normal and tenotonized muscles. In both cases an increase in temperature resulted in a shortening of the twitch time to peak, but in the unoperated muscle this was accompanied by an increase in the twitch tension (cf. cat soleus, Buller, Ranatunga & Smith, 1968) whereas those muscles which had been tenotomized for more than 40 days exhibited a fall in both twitch and tetanus tension.
3
MOTOR UNITS IN TENOTOMIZED RABBIT SOLEUS
Aseptic tenotomies were performed under light pentobarbitone anaesthesia supplemented by 1 % procaine injected subcutaneously around the ankle joint. All the tendons around the right ankle were sectioned, care being taken to avoid damage to the main blood vessels and nerves. Following recovery the animals were kept in pens on a solid floor for a week before being transferred to wire cages. A
B
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Days Days in of twitch Fig. 1. The time course the changes (A) tension, (B) twitch-tetanus ratio, (C) time to peak and (D) time to half relaxation, of rabbit soleus following tenotomy. The continuous horizontal lines represent the mean value and the interrupted lines the mean + S.D. of the fifteen control muscles. Each point represents one tenotomized muscle, the open circles show the four muscles in which motor units were examined.
The terminal experiments were performed on twenty-six animals between 4 and 62 days after tenotomy. Before the animal was anaesthetized an attempt was made to determine if there was any significant tendon regeneration by observing the locomotion of the animals and feeling through the skin for any stretching of the leg muscles when the foot was flexed. In only two cases was it felt that there might have been significant tendon regrowth, and both of these showed less muscular atrophy than might have been expected. These muscles were not used for the motor unit studies. RESULTS
1. The effects of tenotomy upon whole muscle contraction characteristics The tension developed in response to maximal stimulation of the motor nerve fell rapidly in the first 20 days following tenotomy (Fig. 1 A). The tetanus tension declined faster than that of the twitch, resulting in a progressive increase of the twitch-tetanus ratio with time after tenotomy (Fig. 1 B). (The twitch-tetanus ratios in Fig. 1 B and Table 1 are the maximum values recorded. These seldom occurred at the muscle optimum length for either twitches or tetani and so do not correspond to the ratios of the twitch and tetanus tensions in Table 1.) The tetanic response of muscles which had been tenotomized for over 40 days often fell to a plateau after reaching an initial peak of tension (Fig. 2). This was accompanied by a reduction in the amplitude of the electromyogram recorded from the surface of 1
1-2
4
J. BAGUST the muscle suggesting the failure of neuromuscular transmission on repetitive stimulation. This interpretation was confirmed by direct stimulation of the muscle after treatment with curare when the fall in tension was abolished (Fig. 2D). (The experiments involving curare were performed in collaboration with D. M. Lewis). No TABLE 1. The mean isometric contraction properties of the fifteen control solei and the eighteen muscles that had been tenotomized for 20 days or longer. The significance of the differences between the mean values for the two groups is shown in the last column, calculated by Student 's t test (n.s. = difference between the means is not significant, P > 0 05)
Twitch tension(N) Tetanus tension(N) Twitch/tetanus Time to peak (msec) Half relaxation time (msec)
Control
Tenotomized
n mean S.D. 15 3-6 0*9 15 16-2 2-0 15 0-21 004 15 50-8 11*3 15 83-3 32*5
n mean S.D. 18 0*5 0-1 18 1.5 04 18 045 0-11 18 46*7 7-2 18 52*8 14*2
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Fig. 2. Isometric myograms recorded from tenotomized rabbit solei. A, single twitch response from a muscle tenotomized for 33 days; B, tetanic response (80 Hz 300 msec) of the same muscle as in A, superimposed upon a single twitch; C, tetanic response (80 Hz 300 msec) of a soleus muscle that had been tenetomized for 57 days; underneath the mechanical record is the whole muscle electromyogram recorded simultaneously. D, titanic response (80 Hz 300 msec) of a 47 day tenotomized soleus, stimulated indirectly through the nerve (lower record) and directly, after curare (upper record).
5 MOTOR UNITS IN TENOTOMIZED RABBIT SOLEUS attempt was made at systematically quantifying the extent of this neuromuscular failure, but it seems probable that it was an important factor in the increased twitchtetanus ratio. In Table 1 the contraction properties of those muscles examined 20 days or more after tenotomy are compared with corresponding values for the control muscles. The effect of tenotomy upon the time course of the muscle twitch contraction was a non-significant decrease in the time to peak (Fig. 1 C) and a progressive increase in the rate of relaxation as measured by the time to half relaxation (Fig. 1 D). Each of the muscles which had been tenotomized for 50 days or more had a time to half relaxation that was less than half that of the mean control value of 83-3 msec. The greater degree of atrophy of soleus compared to its neighbouring fast-twitch muscles reported by previous authors (McMinn & Vrbova, 1962) was confirmed in these experiments. When compared with muscles from five unoperated animals the mean weight (g/kg body wt.) of solei from thirteen animals which had been tenotomized for more than 20 days, was only 48% ( ± 3 %) of the control value for the tenotomized muscle whereas the corresponding solei from the unoperated side of these animals weighed 109 % ( ± 5%) of the control value. The tenotomized gastrocnemi averaged 67 % ( ± 2 %) of the control value, compared to 107 % ( ± 3 %) for the unoperated muscles, and the corresponding values for plantaris were 67 % ( ± 5 %) and 99 % (± 4 %) respectively. There was little evidence for hypertrophy of the contralateral, unoperated, muscle induced by tenotomy of the right leg.
2. The effect of tenotomy upon the properties of rabbit soleus motor units The control population of ninety-five motor units was isolated from fifteen soleus muscles from unoperated rabbits (sample size from three to ten motor units per experiment). Eight-one motor units were also isolated from ,four of the twenty-six tenotomized rabbit solei approximately six weeks (39, 41, 43 and 45 days) after sectioning all the tendons around the right ankle. These muscles are indicated in Fig. 1 by the open symbols. Because it was not possible to measure the degree of neuromuscular fatigue affecting any individual motor unit, comparison of motor unit contractile strengths between tenotomized and control muscles had to be confined to a consideration of twitch tensions. This might have been expected to result in an underestimate of motor unit size due to the damping effect of the series elastic element. However, the mean value for the 95 control motor unit twitch tensions, when expressed as a percentage of the whole muscle twitch tension, was very close to the corresponding value for the tetanus tensions (mean tetanus tension = *01 0/, S.D. 0-52 %, mean twitch tension = 1.00 %, S.D. 0-54 %/ t = 0.2), suggesting that the errors caused by using twitch tensions to estimate motor unit size were slight. Following tenotomy the mean motor unit twitch tension fell to less than 10% of the mean value for the control units (Table 2). In order to allow comparison of the control motor units with those from the tenotomized muscles the histograms of Fig. 3 have been plotted with each motor unit tension expressed as a percentage of the corresponding whole muscle tension. When treated in this manner no significant difference was found between motor units from the control and tenotomized muscles in either their mean values or in their distributions (Table 2).
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7 MOTOR UNITS IN TENOTOMIZED RABBIT SOLEUS The effect of tenotomy upon the time course of the motor unit twitch contractions was a shortening of both the mean time to peak and time to half relaxation but the histograms of Fig. 3 show that this was not a uniform speeding up of the motor units. The range of motor unit contraction and half relaxation times in the control muscles 25
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was wide, from 22 to 123 msec for the time to peak, and 22-247 msec for the half relaxation times. In the case of the motor units from the tenotomized muscles the slower end of the population was missing. No unit having a time to peak greater than 70 msec, or a half relaxation time greater than 100 msec was found whereas 35 % of the motor units from the control muscles had contraction times longer than 70 msec. These units contributed 21*4 % to the total population tension. At the other end of the spectrum there was no evidence for an increase in the speed of contraction of the faster motor units; the fastest motor unit examined in the tenotomized muscles (contraction time 27 msec, half relaxation time 36 msec) was slower than the fastest control muscles motor unit.
J. BAGUST Possible relationships between axon conduction velocity and motor unit contraction time or tetanus tension were investigated using the method of least squares. Linear regressions were calculated for the ten control experiments in which more than five motor units were isolated. When each experiment was considered individually the 8
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_0 0 40 80 60 80 Conduction velocity (m/sec) Fig. 4. Mechanical responses of a control rabbit soleus muscle and four motor units from that muscle. At the beginning of each trace is a calibration pulse, and the bar under each represents 100 msec. A, whole muscle twitch; B, whole muscle tetanus (80 Hz 300 msec) superimposed on a single twitch response. C-F, single motor unit twitches. G and H. plot of antidromic axon conduction velocity against (G) motor unit time to peak and (H) tetanus tension of ten motor units from a single muscle. The regression lines were fitted by the method of least squares.
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value of the correlation coefficient (r) exceeded the 5 % significance level (P < 0.05) in eight of the ten cases for the regression of contraction time on conduction velocity, and in five out of ten cases for the regressions of tetanus tension on conduction velocity. Fig. 4 shows plots of motor unit contraction times and tetanus tensions against axon antidromic conduction velocities for the control experiment in which the largest number of motor units were examined. When all ninety-five control motor units were treated as one group the significance of the correlation coefficients increased to the 0.1 % level, (regression of contraction time on conduction velocity r = 0 447, regression of tetanus tension on conduction velocity r = 0-471). Following tenotomy significant values for the correlation coefficient were found for the regression of motor unit time to peak upon conduction velocity in two of the four experiments, and when all eighty-one units were treated as a single group there was a high level of correlation (r = 0-366, P < 0.01). Similar treatment of the regression of
9 MOTOR UNITS IN TENOTOMIZED RABBIT SOLEUS motor unit twitch tension upon conduction velocity revealed no significant correlations in either individual experiments or pooled data. In addition to the changes occurring in the contractile characteristics of the motor units following tenotomy evidence was also found for differences in the conduction properties of the nerve axons supplying motor units from control and tenotomized muscles. The mean antidromic conduction velocity increased from a value of 620 m/sec in the control animals to 74*3 m/sec in the tenotomized animals, a difference of 19*8 % which was significant at the 1 % level (Table 2). This observation was not investigated further. DISCUSSION
The results reported here confirm those of Buller & Lewis (1965) that following teneotomy there is a rapid decrease in the ability of the rabbit soleus muscle to develop tension, and that this is accompanied by a slight shortening of the time to peak of the twitch and a larger reduction in the time to half relaxation. They suggest that these changes might be the result of a more rapid atrophy of the slowly contracting fibres within a muscle containing fibres of different contractile speeds, rather than a change in the speed of the contractile elements themselves. This view receives support from the observation that following tenotomy the soleus muscle atrophies faster than its neighbouring fast twitch muscles (rabbit, McMinn & VrbovA 1962; guinea-pig, Tomanek & Cooper 1972). The wide range of motor unit contraction times found in the normal rabbit soleus, and the apparent loss of all the slower motor units in the tenotomized muscles are quite compatible with the concept of differential atrophy. However, if a significant number of motor units within a muscle ceased to function entirely then the remaining units would each contribute a larger fraction to the total muscle tension. The observed loss of the 35 % of the more slowly contracting units from the normal muscle (contributing 21 % of the muscle tension) would be expected to increase the mean motor unit contribution to the whole muscle tension from 1 00 % in the normal muscle to 1*25 % in the tenotomized muscle, but there was no significant change in the mean motor size as measured by the percentage tension, nor any change in the pattern of distribution of tensions between motor units from the control and tenotomized muscles. It is therefore unlikely that a differential atrophy of motor units is responsibe for the changes in muscle contraction characteristics following tenotomy. The possibility of a differential atrophy of muscle fibres within a motor unit has not been excluded by these experiments. The loss of slowly contracting fibres from motor units composed of fibres widely varying in contractile speeds might produce the observed results. Although no direct recordings of the contractile properties of individual muscle fibres within motor units have been made Edstr6m & Kugelberg (1968) have demonstrated a high degree of histochemical uniformity amongst muscle fibres of the same motor unit in the anterior tibial muscle of the rat, suggestive evidence for mechanical uniformity of muscle fibres within motor units. The increase in the mean antidromic conduction velocity of the motor unit axons 40 days after tenotomy was similar to increases in nerve conduction velocities that have been reported by other workers using rabbits in experiments lasting several weeks (Quillam, 1958; Cragg & Thomas, 1964). It therefore seems most probable that this represents an ageing process and is unrelated to the effects of tenotomy. It is
J. BAGUST possible that the differences in motor unit contractile speeds between the control and tenotomized muscles were also an effect of ageing and unrelated to tenotomy. If the correlation between conduction velocity and motor unit time to peak is taken to indicate a causal relationship the increase in antidromic conduction velocity would be expected to result in an increase in the speed of contraction of the motor units, as was found in these experiments. There is no information available about the effects of age on the contractile speeds of motor units in the rabbit soles, but in the cat soleus (a more homogeneus muscle than the rabbit soleus) the mean motor unit time to peak has been shown to increase rather than decrease between six weeks of age and adult (Bagust, Lewis & Westerman, 1974; Bagust, 1974). These experiments were designed to examine the role of the motor unit in the changes taking place in the rabbit soleus muscle following tenotomy. They have shown that a process of differential atrophy of motor units is unlikely to be the explanation for the observed changes in the time course of the twitch contraction, and a change in the contractile elements themselves remains a possibility. They do not give any indication of the factors altered by tenotomy that might result in such changes, except to show that the effects are widespread, involving not only the speed of muscle contraction, but also neuromuscular transmission and even the effects of temperature changes on the twitch tension, and must be interpreted with caution. 10
I would like to thank Dr D. M. Lewis and Professor A. J. Buller for their help and advice. This work was supported by the Muscular Dystrophy Group of Great Britain and the computer used was purchased by a grant from the Medical Research Council.
REFERENCES BAGUST, J. (1974). Relationships between motor nerve conduction velocities and motor unit contraction characteristics in a slow twitch muscle of the cat. J. Physiol. 238, 269-278. BAGUST, J., LEWIS, D. M. & WESTERMAN, R. A. (1974). The properties of motor units in a fast and a slow twitch muscle during post-natal development in the kitten. J. Phy8iol. 237, 75-90. BULLER, A. J. & LEWIS, D. M. (1963). Factors affecting the differentiation of mammalian fast and slow muscle fibres. In The Effect of Use and Disuse on Neuromuscular Functions, ed. GUTMANN, E. & HNIK, P., pp. 149-159. Prague, Amsterdam: Elsevier. BULLER, A. J. & LEWIS, D. M. (1965). Some observations on the effects of tenotomy in the rabbit. J. Physiol. 178, 326-342. BULLER, A. J., RANATUNGA, K. W. & SMIrH, JANET M. (1968). The influence of temperature on the contractile characteristics of mammalian fast and slow twitch skeletal muscles. J. Physiol. 196, 82P. CRAGG, B. G. & THOMAS, P. K. (1964). The conduction velocity of regenerated peripheral nerve fibres. J. Physiol. 171, 164-175. EDSTROM, L. & KUGELBERG, E. (1968). Histochemical composition distribution of fibres and fatigability of singlemotor units. Anterior tibial muscle of the rat. J. Neurosurg. Psychiat. 31, 424-433. McMINw, R. M. H. & VRBOvA, G. (1962). Morphological changes in red and pale muscles following tenotomy. Nature, Lond. 195, 509. QUILLIAM, T. A. (1958). Growth changes in sensory nerve fibre aggregates undergoing remyelination. J. Anat. 92, 383-398. SALMONS, S. & VRBOVA, G. (1969). The influence of activity on some contractile characteristics of mammalian fast and slow muscles. J. Physiol. 201, 535-549. TOMANEK, R. J. & COOPER, R. R. (1972).Ultrastructural changes in tenotomized fast and slow-twitch muscle fibres. J. Anat. 113, 409-424. VRBOVk, G. (1963). The effect of motoneurone activity on the speed of contraction of striated muscle. J. Physiol. 169, 513-526.
